Monoacetylcurcumin: A new inhibitor of eukaryotic DNA polymerase λ and a new ligand for inhibitor-affinity chromatography

Abstract

We previously reported that a phenolic compound, curcumin (diferuloylmethane), was a selective inhibitor of DNA polymerase λ (pol λ) in vitro [Y. Mizushina, M. Hirota, C. Murakami, T. Ishidoh, S. Kamisuki, N. Shimazaki, M. Takemura, M. Perpelescu, M. Suzuki, H. Yoshida, F. Sugawara, O. Koiwai, K. Sakaguchi, Some anti-chronic inflammatory compounds are DNA polymerase λ-specific inhibitors, Biochem. Pharmacol. 66 (2003) 1935–1944.]. We also found that monoacetylcurcumin ([1 E,4 Z,6 E]-7-(4″-acetoxy-3″-methoxyphenyl)-5-hydroxy-1-(4′-hydroxy-3′-methoxyphenyl)hepta-1,4,6-trien-3-on), a chemically synthesized derivative of curcumin, was a stronger pol λ inhibitor than curcumin, achieving 50% inhibition at a concentration of 3.9 μM. Monoacetylcurcumin did not influence the activities of replicative pols such as α, δ, and ε, and showed no effect even on the activity of pol β, the three-dimensional structure of which is thought to be highly similar to that of pol λ. The compound-induced inhibition of pol λ activity was non-competitive with respect to both the DNA template-primer and the dNTP substrate. Monoacetylcurcumin did not inhibit the activity of the C-terminal catalytic domain of pol λ including the pol β-like core, in which the BRCT motif was deleted. The compound did not influence the activities of prokaryotic pols or other mammalian DNA metabolizing enzymes such as calf primase of pol α, calf terminal deoxynucleotidyl transferase, human telomerase, human immunodeficiency virus type-1 reverse transcriptase, T7 RNA polymerase, T4 polynucleotide kinase, and bovine deoxyribonuclease I. Therefore, we concluded that monoacetylcurcumin is a selective inhibitor of pol λ and could be used as a chromatographic ligand to purify pol λ. We then made a monoacetylcurcumin-conjugated column with epoxy-activated Sepharose 6B. In the column, pol λ of full length was selectively adsorbed and eluted.